A highly sensitive biotin-based probe for small RNA northern blot and its application in dissecting miRNA function in pepper.

Small RNAs play important roles in regulation of plant development and response to various stresses. Northern blot is an important technique in small RNA research. Isotope- and biotin- (or digoxigenin) labeled probes are frequently used in small RNA northern blot. However, isotope-based probe is limited by strict environmental regulation and availability in many places in the world while biotin-based probe is usually suffered from low sensitivity. In this study, we developed a T4 DNA polymerase-based method for incorporation of a cluster of 33 biotin-labeled C in small RNA probe (T4BC33 probe). T4BC33 probe reaches similar sensitivity as 32P-labeled probe in dot blot and small RNA northern blot experiments. Addition of locked nucleic acids in T4BC33 probe further enhanced its sensitivity in detecting low-abundance miRNAs. With newly developed northern blot method, expression of miR6027 and miR6149 family members was validated. Northern blot analysis also confirmed the successful application of virus-based miRNA silencing in pepper, knocking down accumulation of Can-miR6027a and Can-miR6149L. Importantly, further analysis showed that knocking-down Can-miR6027a led to upregulation of a nucleotide binding-leucine rich repeat domain protein coding gene (CaRLb1) and increased immunity against Phytophthora capsici in pepper leaves. Our study provided a highly sensitive and convenient method for sRNA research and identified new targets for genetic improvement of pepper immunity against P. capsici.

Novel tripeptide RKH derived from Akkermansia muciniphila protects against lethal sepsis.

OBJECTIVE: The pathogenesis of sepsis is complex, and the sepsis-induced systemic proinflammatory phase is one of the key drivers of organ failure and consequent mortality. Akkermansia muciniphila (AKK) is recognised as a functional probiotic strain that exerts beneficial effects on the progression of many diseases; however, whether AKK participates in sepsis pathogenesis is still unclear. Here, we evaluated the potential contribution of AKK to lethal sepsis development. DESIGN: Relative abundance of gut microbial AKK in septic patients was evaluated. Cecal ligation and puncture (CLP) surgery and lipopolysaccharide (LPS) injection were employed to establish sepsis in mice. Non-targeted and targeted metabolomics analysis were used for metabolites analysis. RESULTS: We first found that the relative abundance of gut microbial AKK in septic patients was significantly reduced compared with that in non-septic controls. Live AKK supplementation, as well as supplementation with its culture supernatant, remarkably reduced sepsis-induced mortality in sepsis models. Metabolomics analysis and germ-free mouse validation experiments revealed that live AKK was able to generate a novel tripeptide Arg-Lys-His (RKH). RKH exerted protective effects against sepsis-induced death and organ damage. Furthermore, RKH markedly reduced sepsis-induced inflammatory cell activation and proinflammatory factor overproduction. A mechanistic study revealed that RKH could directly bind to Toll-like receptor 4 (TLR4) and block TLR4 signal transduction in immune cells. Finally, we validated the preventive effects of RKH against sepsis-induced systemic inflammation and organ damage in a piglet model. CONCLUSION: We revealed that a novel tripeptide, RKH, derived from live AKK, may act as a novel endogenous antagonist for TLR4. RKH may serve as a novel potential therapeutic approach to combat lethal sepsis after successfully translating its efficacy into clinical practice.

Biosensor-based active ingredient recognition system for screening TNF-α inhibitors from lotus leaves.

Erhuangquzhi granules (EQG) have been clinically proven to be effective in nonalcoholic steatohepatitis (NASH) treatment. However, the active components and molecular mechanisms remain unknown. This study aimed to screen active components targeting tumor necrosis factor α (TNF-α) in EQG for the treatment of NASH by a surface plasmon resonance (SPR) biosensor-based active ingredient recognition system (SPR-AIRS). The amine-coupling method was used to immobilize recombinant TNF-α protein on an SPR chip, the specificity of the TNF-α-immobilized chip was validated, and nine medicinal herbs in EQG were prescreened. Nuciferine (NF), lirinidine (ID), and O-nornuciferine (NNF) from lotus leaves were found and identified as TNF-α ligands by UPLC‒MS/MS, and the affinity constants of NF, ID, and NNF to TNF-α were determined by SPR experiments (Kd = 61.19, 31.02, and 20.71 µM, respectively). NF, ID, and NNF inhibited TNF-α-induced apoptosis in L929 cells, the levels of secreted IL-6 and IL-1ß were reduced, and the phosphorylation of IKKß and IκB was inhibited in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In conclusion, a class of new active small-molecule TNF-α inhibitors was discovered, which also provides a valuable reference for the material basis and mechanism of EQG action in NASH treatment.

All-in-One Theranostic Platform Based on Hollow Microcapsules for Intragastric-Targeting Antiulcer Drug Delivery, CT Imaging, and Synergistically Healing Gastric Ulcer.

Bismuth-containing therapies are suggested as first-line and rescue alternatives for gastric ulcer (GU) treatment and Helicobacter pylori eradication. The current treatment strategy is called quadruple therapy and includes proton pump inhibitors, bismuth, and two broad-band antibiotics. This fact may affect medication compliance, leading to a resistance rate of more than 25% to clarithromycin or metronidazole. To counter this, from the perspective of natural products, an intragastric-targeting all-in-one theranostic platform is established: a drug carrier microcapsule composed of multiple synergistic antiulcer drugs, including bismuth, gallotannin, and antibiotics is obtained (BiG@MCs), and the therapeutic effects of BiG@MCs in rodent models are further evaluated. The results show that the BiG@MCs are spherical with homogeneous particle size (3 ± 0.5 µm) and can be response-released to the acidic environment of the stomach (pH 2.0-3.0), preventing the premature release of the BiG@MCs in physiological conditions. It is worth noting that the bismuth component can be easily identified by computed tomography and other detection instruments, which provide the possibility for drug tracing. In summary, these results indicate that BiG@MCs provide a versatile intragastric-targeting drug delivery platform for GU therapeutics.

Pharmacologic targeting of the P-TEFb complex as a therapeutic strategy for chronic myeloid leukemia.

BACKGROUND: The positive transcription elongation factor b (P-TEFb) kinase activity is involved in the process of transcription. Cyclin-dependent kinase 9 (CDK9), a core component of P-TEFb, regulates the process of transcription elongation, which is associated with differentiation and apoptosis in many cancer types. Wogonin, a natural CDK9 inhibitor isolated from Scutellaria baicalensis. This study aimed to investigate the involved molecular mechanisms of wogonin on anti- chronic myeloid leukemia (CML) cells. MATERIALS AND METHODS: mRNA and protein levels were analysed by RT-qPCR and western blot. Flow cytometry was used to assess cell differentiation and apoptosis. Cell transfection, immunofluorescence analysis and co-immunoprecipitation (co-IP) assays were applied to address the potential regulatory mechanism of wogonin. KU-812 cells xenograft NOD/SCID mice model was used to assess and verify the mechanism in vivo. RESULTS: We reported that the anti-CML effects in K562, KU-812 and primary CML cells induced by wogonin were regulated by P-TEFb complex. We also confirmed the relationship between CDK9 and erythroid differentiation via knockdown the expression of CDK9. For further study the mechanism of erythroid differentiation induced by wogonin, co-IP experiments were used to demonstrate that wogonin increased the binding between GATA-1 and FOG-1 but decreased the binding between GATA-1 and RUNX1, which were depended on P-TEFb. Also, wogonin induced apoptosis and decreased the mRNA and protein levels of MCL-1 in KU-812 cells, which is the downstream of P-TEFb. In vivo studies showed wogonin had good anti-tumor effects in KU-812 xenografts NOD/ SCID mice model and decreased the proportion of human CD45+ cells in spleens of mice. We also verified that wogonin exhibited anti-CML effects through modulating P-TEFb activity in vivo. CONCLUSIONS: Our study indicated a special mechanism involving the regulation of P-TEFb kinase activity in CML cells, providing evidences for further application of wogonin in CML clinical treatment. Video Abstract.

LW-213 induces cell apoptosis in human cutaneous T-cell lymphomas by activating PERK-eIF2α-ATF4-CHOP axis.

Cutaneous T-cell lymphoma (CTCL) is characterized by a heterogeneous group of extranodal non-Hodgkin lymphomas, in which monoclonal T lymphocytes infiltrate the skin. LW-213, a derivative of wogonin, was found to induce cell apoptosis in chronic myeloid leukemia (CML). In this study, we investigated the effects of LW-213 on CTCL cells and the underlying mechanisms. We showed that LW-213 (1-25 µM) dose-dependently inhibited human CTCL cell lines (Hut-102, Hut-78, MyLa, and HH) with IC50 values of around 10 µM, meanwhile it potently inhibited primary leukemia cells derived from peripheral blood of T-cell lymphoma patients. We revealed that LW-213-induced apoptosis was accompanied by ROS formation and the release of calcium from endoplasmic reticulum (ER) through IP3R-1channel. LW-213 selectively activated CHOP and induced apoptosis in Hut-102 cells via activating PERK-eIF2α-ATF4 pathway. Interestingly, the degree of apoptosis and expression of ER stress-related proteins were alleviated in the presence of either N-acetyl cysteine (NAC), an ROS scavenger, or 2-aminoethyl diphenylborinate (2-APB), an IP3R-1 inhibitor, implicating ROS/calcium-dependent ER stress in LW-213-induced apoptosis. In NOD/SCID mice bearing Hut-102 cell line xenografts, administration of LW-213 (10 mg/kg, ip, every other day for 4 weeks) markedly inhibited the growth of Hut-102 derived xenografts and prolonged survival. In conclusion, our study provides a new insight into the mechanism of LW-213-induced apoptosis, suggesting the potential of LW-213 as a promising agent against CTCL.

Characterization of Coprecipitates of As(III) and Fe(II) in the Presence of Phyllosilicate Nanoparticles.

Phyllosilicate nanoparticles play an important role in regulating the biogeochemical processes of Fe(II) and As(III) in paddy soils due to their high mobility and activity. In the present work, two prepared muscovite nanoparticles with different sizes (LNPs and SNPs) were used to investigate the effect of the size of phyllosilicate nanoparticles on the coprecipitation of Fe(II) and As(III) during oxidation process. The results showed that muscovite nanoparticles could significantly promote the removal of Fe(II) and As(III) during coprecipitation process. The formation of crystalline iron oxide and oxidation of As(III) tended to be suppressed by the two muscovite nanoparticles, and the suppression increased as muscovite nanoparticle size decrease. The findings of this study provide a contribution to understanding the roles of the natural phyllosilicate nanoparticles in regulating the biogeochemical processes of Fe and As elements in polluted paddy soils.

LW-213, a newly synthesized flavonoid, induces G2/M phase arrest and apoptosis in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell neoplasm characterized by an uncontrolled proliferation of moderately and well differentiated cells of the granulocytic lineage. LW-213, a newly synthesized flavonoid compound, was found to exert antitumor effects against breast cancer through inducing G2/M phase arrest. We investigated whether LW-213 exerted anti-CML effects and the underlying mechanisms. We showed that LW-213 inhibited the growth of human CML cell lines K562 and imatinid-resistant K562 (K562r) in dose- and time-dependent manners with IC50 values at the low µmol/L levels. LW-213 (5, 10, 15 µM) caused G2/M phase arrest of K562 and K562r cells via reducing the activity of G2/M phase transition-related proteins Cyclin B1/CDC2 complex. LW-213 treatment induced apoptosis of K562 and K562r cells via inhibiting the expression of CDK9 through lysosome degradation, thus leading to the suppression of RNAPII phosphorylation, down-regulation of a short-lived anti-apoptic protein MCL-1. The lysosome inhibitor, NH4Cl, could reverse the anti-CML effects of LW-213 including CDK9 degradation and apoptosis. LW-213 treatment also degraded the downstream proteins of BCR-ABL1, such as oncoproteins AKT, STAT3/5 in CML cells, which was blocked by NH4Cl. In primary CML cells and CD34+ stem cells, LW-213 maintained its pro-apoptotic activity. In a K562 cells-bearing mice model, administration of LW-213 (2.5, 5.0 mg/kg, ip, every other day for 4 weeks) dose-dependently prolonged the survival duration, and significantly suppressed huCD45+ cell infiltration and expression of MCL-1 in spleens. Taken together, our results demonstrate that LW-213 may be an efficient agent for CML treatment.

Mitotic catastrophe and p53-dependent senescence induction in T-cell malignancies exposed to nonlethal dosage of GL-V9.

Mitotic catastrophe of cancer cells induced by drugs is characterized by low dosage and low toxicity, representing a significant advantage in the cancer treatment. Effective therapeutic options are limited for T-cell malignancies patients who are still treated by high-dose multiagent chemotherapy, potentially followed by hematopoietic stem cell transplantation, highlighting the urgency for identification of more effective anti-T-cell malignancies drugs. The use of antineoplastic drugs which induced tumor cell mitotic catastrophe would be a new strategy for cancer therapy. Nevertheless, there is still no effective mitotic catastrophe agent in T-cell malignancies. Our study showed that nonlethal dosage (ND) of GL-V9 (5-hydroxy-8-methoxy-2-phenyl-7-(4-(pyrrolidin-1-yl) butoxy) 4 H-chromen-4-one) (2 µM), a potential anticancer drug, not only attenuated cell growth and survival, but also arrested the cell cycle in G2/M phase and induced multipolar spindles, nuclear alterations (micronucleation and multinucleation), which are the most prominent morphological characteristics of mitotic catastrophe, in T-cell malignancies cell lines including Jurkat, HuT-102, and HuT-78. Moreover, ND GL-V9 could trigger DNA damage, and significantly influence several mitosis-associated proteins. Besides, results showed that ND GL-V9 increased the activity of senescence-associated ß-galactosidase (SA-ß-Gal) following the induction of mitotic catastrophe in Jurkat and HuT-102 cells with intact p53, while causing apoptosis in p53-deficient HuT-78 cells. We concluded that the anti-T-cell malignancies effects of ND GL-V9 and clarified the precise regulation in the process of mitosis under the action of GL-V9 in T-cell malignancies. Our data provided new evidence for the study of T-cell malignancies treatment associated with mitotic catastrophe and cellular senescence induction.

Microwave-assisted simultaneous extraction of luteolin and apigenin from tree peony pod and evaluation of its antioxidant activity.

An efficient microwave-assisted extraction (MAE) technique was employed in simultaneous extraction of luteolin and apigenin from tree peony pod. The MAE procedure was optimized using response surface methodology (RSM) and compared with other conventional extraction techniques of macerate extraction (ME) and heat reflux extraction (HRE). The optimal conditions of MAE were as follows: employing 70% ethanol volume fraction as solvent, soaking time of 4 h, liquid-solid ratio of 10 (mL/g), microwave irradiation power of 265 W, microwave irradiation time of 9.6 min, and 3 extraction cycles. Under the optimal conditions, 151 µg/g luteolin and 104 µg/g apigenin were extracted from the tree peony pod. Compared with ME and HRE, MAE gave the highest extraction efficiency. The antioxidant activities of the extracts obtained by MAE, ME, and HRE were evaluated using a 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) free radical-scavenging assay, a ferric reducing antioxidant power assay (FRAP), and a reducing power assay. Meanwhile, the structural changes of the unprocessed and processed tree peony pod samples were analyzed by scanning electron microscopy.

LW-213, a derivative of wogonin, triggers reticulophagy-mediated cell death in NSCLC via lysosomal damage combined with NPC1 inhibition.

BACKGROUND: Maintaining intracellular equilibrium is essential for the viability of tumor cells, which tend to be particularly vulnerable to environmental stressors. Consequently, targeting the disruption of this homeostasis offers a promising approach for oncological treatments. LW-213, a novel derivative of wogonin, effectively induces apoptosis in cancer cells by initiating endoplasmic reticulum (ER) stress, although the precise molecular pathways involved remain intricate and multifaceted. PURPOSE: This research aimed to explore how LW-213 prompts apoptosis in non-small cell lung cancer (NSCLC) cells and to clarify the detailed mechanisms that govern this process. METHODS: Various NSCLC cell lines were utilized to delineate the apoptotic effects induced by LW-213. Advanced methodologies, including RNA sequencing (RNA-seq), Western blotting (WB), immunofluorescence (IF), immunoprecipitation (IP), flow cytometry (Fc), real-time quantitative polymerase chain reaction (RT-qPCR), and electron microscopy, were employed to investigate the underlying molecular interactions. The efficacy and mechanistic action of LW-213 were also assessed in a xenograft model using nude mice. RESULTS: We demonstrated that LW-213, a small molecule cationic amphiphilic drug (CAD), inhibited Niemann-Pick C1 (NPC1) function and induced lysosomal membrane damage, thereby activating the phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway. This activation promoted cholesterol transport from the ER to the lysosome, perpetuating a cholesterol-deficient state in the ER, including massive exocytosis of Ca2+ and activation of FAM134B-mediated reticulophagy. Ultimately, excessive reticulophagy induced lethal ER stress. CONCLUSIONS: In summary, our study elucidates an organelle domino reaction initiated by lysosome damage and a series of self-rescue mechanisms that eventually lead to irreversible lethal effects, revealing a potential drug intervention strategy.

GL-V9 synergizes with oxaliplatin of colorectal cancer via Wee1 degradation mediated by HSP90 inhibition.

OBJECTIVES: GL-V9 exhibited anti-tumour effects on various types of tumours. This study aimed to verify if GL-V9 synergized with oxaliplatin in suppressing colorectal cancer (CRC) and to explore the synergistic mechanism. METHODS: The synergy effect was tested by MTT assays and the mechanism was examined by comet assay, western blotting and immunohistochemistry (IHC). Xenograft model was constructed to substantiated the synergy effect and its mechanism in vivo. RESULTS: GL-V9 was verified to enhance the DNA damage effect of oxaliplatin, so as to synergistically suppress colon cancer cells in vitro and in vivo. In HCT-116 cells, GL-V9 accelerated the degradation of Wee1 and induced the abrogation of cell cycle arrest and mis-entry into mitosis, bypassing the DNA damage response caused by oxaliplatin. Our findings suggested that GL-V9 binding to HSP90 was responsible for the degradation of Wee1 and the vulnerability of colon cancer cells to oxaliplatin. Functionally, overexpression of either HSP90 or WEE1 annulled the synergistic effect of GL-V9 and oxaliplatin. CONCLUSIONS: Collectively, our findings revealed that GL-V9 synergized with oxaliplatin to suppress CRC and displayed a promising strategy to improve the efficacy of oxaliplatin.

Paclitaxel combined with Compound K inducing pyroptosis of non-small cell lung cancer cells by regulating Treg/Th17 balance.

BACKGROUND: Immune checkpoint inhibitors, which have attracted much attention in recent years, have achieved good efficacy, but their use is limited by the high incidence of acquired drug resistance. Therefore, there is an urgent need to develop new immunotherapy drugs. Compound taxus chinensis capsule (CTC) is an oral paclitaxel compound drug, clinical results showed it can change the number of regulatory T cells and T helper cell 17 in peripheral blood. Regulating the balance between regulatory T cells and T helper cell 17 is considered to be an effective anticancer strategy. Paclitaxel and ginsenoside metabolite compound K are the main immunomodulatory components, it is not clear that paclitaxel combined with compound K can inhibit tumor development by regulating the balance between regulatory T cell and T helper cell 17. METHODS: MTT, EdU proliferation and plate colony formation assay were used to determine the concentration of paclitaxel and compound K. AnnexinV-FITC/PI staining, ELISA, Western Blot assay, Flow Cytometry and Immunofluorescence were used to investigate the effect of paclitaxel combined with compound K on Lewis cell cultured alone or co-cultured with splenic lymphocyte. Finally, transplanted tumor C57BL/6 mice model was constructed to investigate the anti-cancer effect in vivo. RESULTS: According to the results of MTT, EdU proliferation and plate colony formation assay, paclitaxel (10 nM) and compound K (60 µM) was used to explore the mechanism. The results of Flow Cytometry demonstrated that paclitaxel combined with compound K increased the number of T helper cell 17 and decreased the number of regulatory T cells, which induced pyroptosis of cancer cells. The balance was mediated by the JAK-STAT pathway according to the results of Western Blot and Immunofluorescence. Finally, the in vivo results showed that paclitaxel combined with compound K significantly inhibit the progression of lung cancer. CONCLUSIONS: In this study, we found that paclitaxel combined with compound K can activate CD8+ T cells and induce pyroptosis of tumor cells by regulating the balance between regulatory T cells and T helper cell 17. These results demonstrated that this is a feasible treatment strategy for lung cancer.

Identification of the chemical constituents in Cullen corylifolium ethanolic extract by LC-MS/MS and GC-MS.

Cullen corylifolium has been used to treat bronchial asthma, vitiligo, nephritis and leucopenia from ancient times in China. However, its chemical composition has not been fully understood. In this study, both LC-MS and GC-MS were used to identify the chemical composition in C. corylifolium. Consequently, 31 compounds were qualified by LC-MS/MS, 8 representative compounds of which were quantified, 62 compounds including hydrocarbons, terpenoids and coumarins were identified by GC-MS. Among a total of 93 compounds, 32 compounds (8 by LC-MS/MS and 24 by GC-MS) are undocumented for this herb. Our results will provide a crucial foundation for further studies related to its pharmaceutical activities and quality control.

LW-213 induces immunogenic tumor cell death via ER stress mediated by lysosomal TRPML1.

Dying tumor cells release biological signals that exhibit antigenicity, activate cytotoxic T lymphocytes, and induce immunogenic cell death (ICD), playing a key role in immune surveillance. We demonstrate that the flavonoid LW-213 activates endoplasmic reticulum stress (ERS) in different tumor cells and that the lysosomal calcium channel TRPML1 mediates the ERS process in human cellular lymphoma Hut-102 cells. Apoptotic tumor cells induced by ERS often possess immunogenicity. Tumor cells treated with LW-213 exhibit damage-associated molecular patterns (DAMPs), including calreticulin translocation to the plasma membrane and extracellular release of ATP and HMGB1. When co-cultured with antigen-presenting cells (APCs), LW-213-treated tumor cells activated APCs. Two groups of C57BL/6J mice were inoculated with Lewis cells: a "vaccine group", which demonstrated that LW-213-treated tumor cells promote the maturation of dendritic cells and increase CD8+ T cells infiltration in the tumor microenvironment and a "pharmacodynamic group", treated with a combination of LW-213 and PD1/PD-L1 inhibitor (BMS-1), which reduced tumor growth and significantly prolonged the survival time of mice in the "pharmacodynamic group". Therefore, LW-213 can be developed as a novel ICD inducer, providing a new concept for antitumor immunotherapy.

Targeting lysosomal HSP70 induces acid sphingomyelinase-mediated disturbance of lipid metabolism and leads to cell death in T cell malignancies.

BACKGROUND: T cell malignancies proliferate vigorously, are highly dependent on lysosomal function, with limited therapeutic options. Deregulation of lysosomal structure and function has been confirmed to be a key role in the treatment of hematologic malignant disease. METHODS: Cell counting kit 8 and Annexin V/PI staining were used to assess the cell viability and apoptosis rate. Flow cytometry, liquid chromatography mass spectrometry, immunofluorescence and western blot were performed to detect the effect on lysosomes. Drug affinity responsive target stability, molecular docking and cellular thermal shift assay were employed to confirm the target protein of V8 on lysosomes. A xenograft model was constructed in NOD/SCID mice to assess the effect and mechanism. RESULTS: V8, a new lysosomotropic compound, could be rapidly trapped by lysosomes and accumulation in lysosomes, contributing to lysosomal-dependent cell death by evoking lysosomal membrane permeabilization (LMP), accompanied with disrupted lysosome and autophagic flux. Mechanistically, heat shock protein 70 (HSP70) was identified as the binding target of V8 in lysosome. As a downstream effect of targeting HSP70, enzymatic activity of acid sphingomyelinase (ASM) was inhibited, which induced disturbance of lipid metabolism, instability of lysosomal membrane, and leakage of cathepsin B and D, leading to LMP-mediated cell death. In vivo study showed V8 well controlled the growth of the tumour and confirmed lysosomal cell death induced by V8. CONCLUSIONS: Collectively, this study suggests targeting lysosomal HSP70-ASM axis by V8 illustrates the great value of drug therapy for T cell malignancies and the unlimited potential of lysosomal targeting for cancer therapy.

A New Biotin Labeling and High-Molecular-Weight RNA Northern Method and Its Application in Viral RNA Detection.

Viruses cause severe crop losses. Studying the interaction between viruses and plants is very important for development of control measures. Northern blot is a well-accepted but very challenging technique to monitor the infection of viruses. Here, we modified the high-molecular-weight (hmw)RNA Northern blot experiment process, utilizing vertical electrophoresis to separate the RNA with denatured agarose gel. This protocol is compatible with regular equipment for Western blots and small RNA Northern blots and requires less input of total RNA. A new method to label the probe with biotin was also developed, which requires commonly used T4 DNA polymerase and detects viral RNA with high sensitivity. These new protocols made hmwRNA Northern blot cost-effective and easy-to-operate, very suitable for studying virus-host interactions.

Cotyledon loss of Astragalus membranaceus hindered seedling establishment through mineral element reallocation and carbohydrate depletion.

Tissue loss of plants caused by herbivores is very common in nature. As the storage and first photosynthetic organ, the loss of cotyledon severely impacts dicot seedling establishment and the subsequent growth. However, it is still not clear how plants adjust their metabolic strategy in response to cotyledon loss. In this study, we employed ICP-OES, GC and LC-MS to examine the effects of cotyledon removal (RC1: remove one cotyledon, RC2: remove two cotyledon) on mineral element distribution and metabolite changes in a traditional Chinese herbal plant, Astragalus membranaceus. The results showed that cotyledon removal had a greater effect on shoot than root growth. Specifically, RC2 revealed a more serious impact on shoot growth than RC1. Microelement Mn and Na in shoot increased more in RC2 than RC1. Macroelement K and microelement B in root increased in RC2. The metabolite results in shoot showed that sugars related to galactose metabolism reduced while amino acids significantly increased in RC2. In root, sugars related to fructose and mannose metabolism decreased in both RC1 and RC2 while most flavonoids increased in RC2. It can be concluded that cotyledon removal triggered different metabolic strategies in both root and shoot. In shoot, more Mn was absorbed to improve the lowered photosynthetic efficiency. Meanwhile, increased Na may have promoted carbohydrate consumption and amino acid synthesis, thereby maintaining shoot growth. In root, K and B participation in cell division and expansion increased, as well as the delivery and metabolism of carbohydrates, to maintain root system growth.

Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218.

The integrity of lysosomes is of vital importance to survival of tumor cells. We demonstrated that LW-218, a synthetic flavonoid, induced rapid lysosomal enlargement accompanied with lysosomal membrane permeabilization in hematological malignancy. LW-218-induced lysosomal damage and lysosome-dependent cell death were mediated by cathepsin D, as the lysosomal damage and cell apoptosis could be suppressed by depletion of cathepsin D or lysosome alkalization agents, which can alter the activity of cathepsins. Lysophagy, was initiated for cell self-rescue after LW-218 treatment and correlated with calcium release and nuclei translocation of transcription factor EB. LW-218 treatment enhanced the expression of autophagy-related genes which could be inhibited by intracellular calcium chelator. Sustained exposure to LW-218 exhausted the lysosomal capacity so as to repress the normal autophagy. LW-218-induced enlargement and damage of lysosomes were triggered by abnormal cholesterol deposition on lysosome membrane which caused by interaction between LW-218 and NPC intracellular cholesterol transporter 1. Moreover, LW-218 inhibited the leukemia cell growth in vivo. Thus, the necessary impact of integral lysosomal function in cell rescue and death were illustrated.